Open AccessVol 9 No 5 Research article Catechol-O-methyltransferase gene haplotypes in Mexican and Spanish patients with fibromyalgia Gilberto Vargas-Alarcón1, José-Manuel Fragoso1, Davi
Trang 1Open Access
Vol 9 No 5
Research article
Catechol-O-methyltransferase gene haplotypes in Mexican and Spanish patients with fibromyalgia
Gilberto Vargas-Alarcón1, José-Manuel Fragoso1, David Cruz-Robles1, Angélica Vargas1,
Alfonso Vargas1, José-Ignacio Lao-Villadóniga2, Ferrán García-Fructuoso3, Manuel Ramos-Kuri4, Fernando Hernández4, Rashidi Springall1, Rafael Bojalil1, Maite Vallejo1 and Manuel Martínez-Lavín1
1 National Institute of Cardiology, Juan Badiano 1, Mexico City 14080, Mexico
2 Dr Echevarne Laboratory, Provenza 312, Barcelona E08037, Spain
3 CIMA Clinic, Manuel Girona 33, Barcelona E08034, Spain
4 Panamerican University, Donatelo 59, Mexico City 03920, Mexico
Corresponding author: Manuel Martínez-Lavín, mmlavin@infosel.net.mx
Received: 19 Apr 2007 Revisions requested: 25 May 2007 Revisions received: 20 Jun 2007 Accepted: 26 Oct 2007 Published: 26 Oct 2007
Arthritis Research & Therapy 2007, 9:R110 (doi:10.1186/ar2316)
This article is online at: http://arthritis-research.com/content/9/5/R110
© 2007 Vargas-Alarcón et al.; licensee BioMed Central Ltd
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Autonomic dysfunction is frequent in patients with fibromyalgia
(FM) Heart rate variability analyses have demonstrated signs of
ongoing sympathetic hyperactivity Catecholamines are
sympathetic neurotransmitters Catechol-O-methyltransferase
(COMT), an enzyme, is the major catecholamine-clearing
pathway There are several single-nucleotide polymorphisms
(SNPs) in the COMT gene associated with the different
catecholamine-clearing abilities of the COMT enzyme These
SNPs are in linkage disequilibrium and segregate as
'haplotypes' Healthy females with a particular COMT gene
haplotype (ACCG) producing a defective enzyme are more
sensitive to painful stimuli The objective of our study was to
define whether women with FM, from two different countries
(Mexico and Spain), have the COMT gene haplotypes that have
been previously associated with greater sensitivity to pain All
the individuals in the study were female Fifty-seven Mexican
patients and 78 Spanish patients were compared with their
respective healthy control groups All participants filled out the Fibromyalgia Impact Questionnaire (FIQ) Six COMT SNPs (rs2097903, rs6269, rs4633, rs4818, rs4680, and rs165599) were genotyped from peripheral blood DNA In Spanish patients, there was a significant association between three SNPs (rs6269, rs4818, and rs4680) and the presence of FM when compared with healthy controls Moreover, in Spanish patients with the 'high pain sensitivity' haplotype (ACCG), the disease, as assessed by the FIQ, was more severe By contrast, Mexican patients displayed only a weak association between rs6269 and rs165599, and some FIQ subscales In our group
of Spanish patients, there was an association between FM and the COMT haplotype previously associated with high pain sensitivity This association was not observed in Mexican patients Studies with a larger sample size are needed in order
to verify or amend these preliminary results
Introduction
Fibromyalgia (FM) is a frequent illness Epidemiological
stud-ies from different parts of the world have shown a prevalence
rate of 2% to 3% in the general population Approximately
90% of affected individuals are female [1] The severity of the
illness diminishes the quality of life of afflicted persons [2] and
imposes a heavy economic burden on society [3] Therefore,
FM can be considered a major health problem among contem-porary women
Different groups of investigators have shown that autonomic dysfunction is frequent in patients with FM Heart rate variabil-ity analyses have demonstrated signs of ongoing sympathetic hyperactivity This hyperactivity is accompanied by a blunted COMT = catechol-O-methyltransferase; FIQ = Fibromyalgia Impact Questionnaire; FM = fibromyalgia; HLA = human leukocyte antigen; LD = linkage disequilibrium; SNP = single-nucleotide polymorphism.
Trang 2sympathetic response to different stressors It has been
pro-posed that this autonomic dysfunction explains the
multifac-eted complaints of FM [4] The defining characteristics of FM
(namely, widespread pain and allodynia) could be explained by
a pathogenesis known as 'sympathetically maintained pain'
[5]
Naturally occurring sympathetic neurotransmitters are
cate-cholamines known as norepinephrine, epinephrine, and
dopamine All three substances act within the central nervous
system Norepinephrine acts also in peripheral postganglionic
nerve endings and exerts local effects in close proximity to the
area where it is released, whereas epinephrine is the
circulat-ing hormone of the adrenal medulla and influences processes
throughout the body
The major systemic transformation of catecholamines is
cata-lyzed by the catechol-O-methyltransferase (COMT) enzyme
The COMT gene is located in region 22q11.1 to 22q11.2 of
chromosome 22 There are different single-nucleotide
poly-morphisms (SNPs) in the COMT gene which induce important
functional alterations of the enzyme The best-studied SNP
(rs4680) occurs in codon 158 with valine-to-methionine
tran-sition (Val-158-Met) The Val-158-Val genotype gives rise to
an effective enzyme, whereas the Met-158-Met genotype
pro-duces a defective enzyme, which is incapable of effectively
clearing catecholamines from the system [6]
Zubieta and colleagues [7] demonstrated that healthy
individ-uals with the COMT Val-158-Val genotype are pain-resistant
The opposite occurs in people with the Met-158-Met
geno-type Gursoy and colleagues [8], of Turkey, reported an
asso-ciation between FM and the Val-158-Met COMT
polymorphism García-Fructuoso and colleagues [9], of Spain,
described that FM patients with the Met-158-Met genotype
have a more severe form of the disease when compared with
affected individuals with the Val-158-Val genotype
More meticulous genetic studies demonstrated that COMT
functional properties are only marginally dependent on the
Val-158-Met transition There are other SNPs in the COMT gene
(rs6269, rs4633, and rs4818) in linkage disequilibrium (LD)
with the Val-158-Met variation By combining these SNPs,
Diatchenko and colleagues [10] were able to identify frequent
COMT gene haplotypes strongly associated with sensitivity to
experimental pain and induction of a more defective COMT
enzyme Healthy females with the 'high pain sensitivity
haplo-type' have a COMT enzyme 11 times less efficient than
per-sons with the 'low pain sensitivity haplotype' Diatchenko and
colleagues [10] also demonstrated that COMT inhibition in
rats results in a dramatic increase in sensitivity to pain Low
COMT activity may increase pain sensitivity by activating beta
(2/3)-adrenergic receptors The objective of our investigation
was to define whether FM patients from two different countries
(Mexico and Spain) have COMT alleles associated with increased susceptibility to pain in healthy individuals
Materials and methods
Patients
All the subjects studied were women The criterion for inclu-sion was to be diagnosed with FM as per the guidelines of the American College of Rheumatology and to be free of concur-rent rheumatic disease These patients were sourced from dif-ferent private rheumatology practices in Mexico and Spain Mexican controls were women who considered themselves to
be healthy and who denied having chronic pain Twenty-seven
of them were paramedical personnel and 6 were relatives of paramedical personnel but were not related among them-selves Spanish controls were blood donors who denied hav-ing chronic pain Patients and controls were matched by gender and age Informed consent was obtained from all par-ticipants, and the Human Research Committee of the National Institute of Cardiology of Mexico approved the study
Both patients and controls filled out a validated Spanish trans-lation of the Fibromyalgia Impact Questionnaire (FIQ) [11] This is an instrument designed to numerically define the overall impact of FM across many dimensions (for example, function, pain level, fatigue, sleep disturbance, and psychological dis-tress) [12]
Genotyping
Genomic DNA from whole blood containing EDTA (ethylene-diaminetetraacetic acid) was extracted by standard tech-niques [13] Six COMT SNPs (rs2097903, rs6269, rs4633, rs4818, rs4680, and rs165599) were genotyped using 5' exo-nuclease TaqMan assays on an ABI Prism 7000 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions The National Center for Biotechnology Information (Bethesda, MD, USA) SNP database was used to assign SNP numbers
Statistical analysis
The χ2 test was used to evaluate the Hardy-Weinberg equilib-rium for each polymorphism Statistical analysis was carried out with Stata 8.0 for Windows software (College Station, TX, USA) During the exploratory analysis, our numerical data showed a non-Gaussian distribution (Shapiro-Wilk test,
p > 0.05) Therefore, Kruskal-Wallis tests were used to
com-pare these variables Data are presented as median and 25th and 75th percentiles Categorical variables were analyzed with χ2 or Fisher tests as required and presented as absolute frequencies and proportions Statistical significance was set
at an alpha level of less than or equal to 0.05 The total FIQ score was correlated with genotypes and haplotypes Mexican and Spanish groups were examined separately Pairwise link-age disequilibrium (LD, D') estimations between polymor-phisms and haplotype reconstruction were performed with Haploview version 3:32 (Broad Institute of Massachusetts
Trang 3Institute of Technology and Harvard University, Cambridge,
MA, USA)
Results
Table 1 shows the demographic data of the populations
stud-ied The two groups of patients had similar age distributions
and FIQ scores However, Mexican controls had higher FIQ
scores when compared with Spanish controls (p < 0.05).
Genotype distribution of all COMT SNPs studied in both
pop-ulations is shown in Table 2 The observed and expected
fre-quencies of the different SNPs in both populations were in
Hardy-Weinberg equilibrium Statistically significant
associa-tions were found between three SNPs (rs6269, rs4818, and
rs4680) and the development of FM in Spaniards In
Mexi-cans, the distribution of the six SNPs did not differ between
patients and controls
Table 3 depicts the correlation between the SNPs and total
FIQ score Again, there was a strong correlation between high
FIQ score and four SNP genotypes (rs6269AA, rs4633CC,
rs4818CC, and rs4680GG) in Spaniards No correlation was
evident in Mexicans
FIQ contains several visual analogue scales In Spaniards,
there was an association between the above-mentioned SNPs
and the visual analogue scale scores for pain fatigue, sleep
disturbance, and morning stiffness In Mexicans, a significant
correlation was found between rs6269 and pain and fatigue
as well as between rs165599 and disability and morning
stiffness
LD analyses between the six SNP markers showed that four of
them – rs6269, rs4633, rs4818, and rs4680 (located in the
central region of the COMT gene) – had strong LDin both
pop-ulations The strongest associations were found between
SNPs rs6269 with rs4680, rs6269 with rs4818, and rs4633
with rs4680 (D' = 0.986, R2 = 0.878) In light of this finding,
we analyzed the most frequent haplotypes in patients and
healthy controls to determine whether some of these
haplo-types could be associated with the risk of developing FM
among the populations in the study There were four frequent
haplotypes in Spaniards (ACCG, ATCA, GCGG, and GTGA)
Haplotype distribution was different in Spanish patients versus
controls (p = 0.006) Patients had higher ACCG and ATCA
frequencies and a lower GTGA frequency Mexicans had three frequent haplotypes (ACCG, ATCA, and GCGG) that were distributed similarly among patients and controls (Table 4) In Spanish patients, the ACCG haplotype was strongly
associ-ated with a high-percentile FIQ score (p = 0.0001) (Table 5).
Discussion
FM strongly aggregates in families and coaggregates with mood disorders [14] Nevertheless, previous attempts to show
a specific genetic defect in FM yielded weak and/or divergent results In a multicase family study, Yunus and colleagues [15] found a feeble association with the human leukocyte antigen (HLA) region The reported relationship to the serotonin
trans-porter gene (5-HTTLPR) [16] is probably linked to comorbid
depression rather than to FM itself [17] Studies on the dopamine D4 exon III repeat polymorphism yield inconsistent results [18]
In view of the sympathetic dysfunction demonstrated in FM, the COMT gene became an attractive genetic target Explora-tory studies by Gursoy and colleagues [8] and García-Fructu-oso and colleagues [9] uncovered an association between the Val-158-Met (rs4680) COMT transition and FM In contrast, Hagen and colleagues [19], using mail questionnaires, found
no association between Val-158-Met transition and chronic musculoskeletal pain Since there was no direct contact with patients, the percentage of people with musculoskeletal pain actually had FM was not established Another report by the same group using the same method described an association between Val-158-Met polymorphism and headache [20]
In the study of Diatchenko and colleagues [10] of healthy female volunteers, SNP rs4818 accounted for 7% of pain sen-sitivity, SNP rs6269 for 6%, and SNP rs4680 (Val-158-Met) showed only a marginal relationship with pain sensitivity Our results in the Spanish population are in line with these consid-erations Such SNPs were associated not only with the diag-nosis of FM, but also with the severity of the illness as assessed by FIQ scores Additionally, we found SNP rs4633
to be associated with FM in Spaniards This SNP, which rep-resents a synonymous variation (does not produce a change
Table 1
Demographic data of the studied populations
All tested individuals were women Mexican controls had a higher Fibromyalgia Impact Questionnaire (FIQ) score when compared with Spanish
controls (p < 0.05) a SD, standard deviation.
Trang 4in amino acid composition), was not found to be associated
with pain sensitivity in the study of Diatchenko and colleagues
[10]
The differences observed between Spanish and Mexican
pop-ulations were an unexpected result of our study We view two
possible explanations for such a discrepancy
1 Population differences The Mexican population has been
shown to be one of the most vigorous genetic admixtures, with
genes of Caucasian, Amerindian, and African origin A parental
group represented by Spaniards has a more homogeneous
background Vargas-Alarcón and colleagues [21] analyzed the
HLA allele distribution in 69 populations around the world,
including Mexicans and Spaniards Genetic distances with corresponding analyses showed important differences between these two groups [21] Previously, it has been recog-nized that, in the presence of COMT alleles, there will be eth-nic variations Such variations were found not only among the general population, but also in association with disease [6]
2 Sampling error and other stratifications Control groups were different Mexicans were paramedical personnel and other women who considered themselves to be healthy Para-medical personnel are not the most representative of all peo-ple without FM in the population from which the FM cases were obtained Mexican controls had higher FIQ scores than their blood-donor Spanish counterparts We consider that this
Genotype distribution of the six catechol-O-methyltransferase single-nucleotide polymorphisms in patients and healthy controls from Mexico and Spain
rs6269
rs4633
rs4818
rs4680
rs20907
rs16559
a χ 2 test; NS, not significant.
Trang 5Table 3
Comparison of single-nucleotide polymorphisms and Fibromyalgia Impact Questionnaire scores in patients from Mexico and Spain
Genotypes 25th
percentile
percentile
P value 25th
percentile
percentile
P value
rs6269
rs4633
rs4818
rs4680
Only single-nucleotide polymorphisms with significant associations in Spaniards are shown In Mexicans, none was associated with the
Fibromyalgia Impact Questionnaire.
Table 4
Haplotype frequencies in patients and healthy controls from Mexico and Spain
Haplotypes
Each frequency is calculated by dividing the occurrence of a given haplotype by the total number of haplotypes in that particular group (haplotype total = number of individuals × 2) NS, not significant.
Trang 6bias is unlikely to explain our diverging results Even after the
Mexican controls with the highest FIQ score were excluded
from our calculations, differences persisted Furthermore,
although Mexican and Spanish patients had similar FIQ
scores, they displayed a significantly different distribution in
COMT SNP rs6269, rs16559, and rs4818
Several reports suggest that the collective grouping of SNPs
in haplotypes has a stronger association with the assessed
phenotype In our study, COMT haplotype analysis showed a
strong association between ACCG and ATCA and the risk of
developing FM in Spanish individuals Additionally, the ACCG
haplotype was associated with a high FIQ score in Spanish
patients Previously, this haplotype has been designated as
'high pain sensitivity' due to the fact that healthy women with
this particular genetic make-up are much more sensitive to
painful stimuli Our results suggest that an ACCG haplotype
could be an important susceptibility marker for FM in the
Span-ish population
Conclusion
Our results show an association between FM and the COMT
pain sensitivity SNPs in Spaniards By contrast, the Mexican
population displayed only a weak association between the two
SNPs and isolated FM symptoms This phenomenon suggests
that there are population variations in susceptibility to
develop-ing FM which are related to the COMT gene polymorphism
Studies with a larger sample size are needed in order to verify
or amend these preliminary results
Competing interests
The authors declare that they have no competing interests
Authors' contributions
GVA, JMF, DCR, MRK, and FH carried out the molecular
genetic studies FGF and JILV recruited and studied the
Span-ish population and purified DNA samples RB and RS purified
DNA samples from Mexico AV and AV performed the clinical
studies on Mexican participants MV carried out the statistical
analyses MML conceived the study, participated in its design
and coordination, and helped to draft the manuscript All authors read and approved the final manuscript
Acknowledgements
We gratefully acknowledge the financial support of the American Fibro-myalgia Syndrome Association (AFSA) This association had no role in study design; in the collection, analysis, and interpretation of data; in the writing of the manuscript; or in the decision to submit the manuscript for publication Teresa de la Canal provided editorial assistance and was paid with the AFSA grant.
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